Recently we discovered a single point mutation in estrogen receptor alpha, replacing lysine 303 with arginine (K303R ERalpha), in about a third of clinical premalignant breast ductal hyperplasias. Transgenic mice expressing the mutation in the mammary gland likewise develop highly proliferative ductal hyperplasias. The mutant ER is hyper-sensitive to estrogen, suggesting that it might drive inappropriate growth and therefore propel these hyperplasias toward malignancy, invasion, and ultimately metastasis. Indeed, we found that the K303R ER mutation is present in 38% of node-negative breast cancers, but in 85% of node-positive breast cancers. The K303R ERalpha mutation is thus the most frequent mutation reported in invasive breast cancer, indicating that it could be a marker of aggressive metastatic behavior. We hypothesize that activation of ERalpha by mutation at the K303 site is a "permissive" event occurring early during breast cancer evolution, and that its hypersensitive phenotype provides a proliferative advantage which could favor tumor progression and metastasis. Because the mutation is seldom if ever found in normal tissue, it could provide a highly specific treatment target. We therefore propose: 1) To determine whether the K303R ERalpha mutation influences the metastatic behavior of breast cancer cells. Sup analysis will be used to sequence the mutation in paired primary and lymph node or distant metastases from breast cancer patients. The role of the ER mutant in metastatic behavior will be examined both in vitro and in xenograft models. 2) To determine whether the mutation alters outcome of hormonal therapies, in specimens from a clinical trial and in a bitransgenic mouse model. 3) To determine how the mutation might be exploited as a target for new therapeutic strategies. Using peptide display technology, we will develop peptide antagonists to ER binding of the coactivator SRC-2, which we have found to be greatly enhanced by the ER mutation, and will assess the ability of these peptide antagonists to inhibit ER function and block the growth of breast cell lines overexpressing the mutant ER.